PROJECT SUMMARY/ABSTRACT: DNA double strand(DSB) breaks are repaired through non-homologous end-joining (NHEJ), microhomology- mediated end-joining (MMEJ), or homologous recombination (HR). These pathways are of central importance in the carcinogenesis of HR deficient cancers and in the response to DNA damaging agents. However, the pathways remain challenging to measure directly apart from using specialized cell lines with stably integrated DNA repair reporter cassettes or by indirectly measuring repair through visualization of pathway factors within irradiation induced foci. We have developed a simple system to measure the usage of all three pathways at a single genomic location using Cas9 to create double strand breaks and next generation sequencing to profile and quantify pathway choice in a pool of cells (DSBR-seq). Principal component analyses using experiments in isogenic cell lines deficient in each pathway are used to classify MMEJ genomic scars separately from NHEJ scars. The system can be used in any live cell to which Cas9 can be delivered, obviating the need for reporter cell lines. We have demonstrated that almost all pathway-specific information can be reduced to just a few dominant types of scars that are characteristic to a genomic locus. Thus, the selected dominant reads can be probed with droplet digital PCR (DSBR-ddPCR). The overall objective of this proposal is to further develop this approach for wide usage in basic DSB repair research. There are also important downstream applications in translational science, such as detection of HR deficient cancers and use in DNA repair inhibitor clinical trials. The central hypothesis is that these approaches can replace reporter cassettes as the preferred method to measure DSB repair and allow for measurement of diverse types of genomic scars. In Aim 1, we will maximize applicability of DSBR-seq through advanced development in human and murine cells and validate the approach through comparisons to reporter cassettes using positive and negative controls that perturb each pathway. In Aim 2, we will develop and validate a simpler readout of pathway capacity through DSBR-ddPCR, which can be broadly used in research laboratories without the need for next-generation sequencing and bioinformatic analyses. Our quantitative milestones are designed to compare DSBR-seq/DSBR-ddPCR directly against cassette reporter assays. These proposed aims would provide a significant contribution due to the potentially broad applicability of the technologies in basic and preclinical research as the study of DSB repair constitutes a large swath of NCI-sponsored research activities.